RNA PROCESSING AND NUCLEAR RIBONUCLEOPROTEIN

描述(改编自申请者摘要)：长期目标 这项研究的目的是为了了解其机制和控制。 真核基因的表达，特别强调它的作用 调控转录后加工的RNA相关蛋白 事件。目前的项目，以果蝇为实验系统， 使用两种一般的实验方法来解决这个问题：(A)分子 主要结构蛋白(HnRNPs)功能的遗传分析 和(B)RNA的超微结构分析 转录、核糖核蛋白(RNP)形成和早期RNA加工 使用米勒染色质铺展方法的新生成绩单事件。 过去三年取得的成果为 对这种普遍存在的蛋白质的功能进行遗传测试，这些蛋白质是 转录时沉积在RNA上，形成hnRNP复合体，该复合体 可能用作后续处理事件的底物。 果蝇有两个单拷贝基因，编码五个相似的基本hnRNP 约40 kDa的蛋白质(因此与哺乳动物的系统非常相似)。 其中一个会发生突变，这样外源hnRNP基因就可以 介绍一种条件启动子及hnRNP蛋白的作用 研究了耗竭。 灵敏的基因分析将允许研究对 转录延长、剪接和多聚腺苷化，所有这些都需要 与hnRNP情结紧密地联系在一起，或者放在hnRNP情结上。实验 还计划引入改变后的基因拷贝，以便发挥作用 不同的蛋白质亚型(通过选择性剪接产生)和不同的 蛋白质中的结构基序(如RNP共有序列)可以 在活的有机体中进行分析。异构体特异性免疫-EM 抗体将指示是否所有形式的蛋白质都在 给定基因的转录本，或者它们是否以特定于基因的方式结合 核糖核酸分子。这种方法应该第一次允许直接的 测试这些无处不在的丰富蛋白质的功能，它们的作用 在前信使核糖核酸酶的代谢方面，人们知之甚少，也颇有争议。 计划进行更多的实验，以继续进行EM超微结构分析 体内的剪接过程。

DESCRIPTION (Adapted from applicant's abstract): The long term goals of the research program are to understand the mechanisms and control of eukaryotic gene expression with particular emphasis on the role of RNA-associated proteins in regulating post-transcriptional processing events. The current project, with Drosophila as the experimental system, uses two general experimental approaches to the problem: (a) molecular genetic analysis of the function of the major structural proteins (hnRNPs) associated with nuclear pre-mRNA, and (b) ultrastructural analysis of RNA transcription, ribonucleoprotein (RNP) formation, and early RNA processing events on nascent transcripts using the Miller chromatin spreading method. Results obtained over the last three years have laid the groundwork for a genetic test of function of this ubiquitous class of proteins, which are deposited on RNA as it is transcribed, forming an hnRNP complex that presumably serves as the substrate for subsequent processing events. Drosophila has two single copy genes encoding five similar basic hnRNP proteins of about 40 kDa, (and thus is quite similar to mammalian systems). One of these will be mutated so that an exogenous hnRNP gene can be introduced on a conditional promoter and the effects of hnRNP protein depletion studied. Sensitive genetic assays will allow study of subtle effects on transcription elongation, splicing and polyadenylation, all of which take place in intimate association with, or on, an hnRNP complex. Experiments are also planned to introduce altered copies of the gene so that the role of various protein isoforms (produced by alternative splicing), and various structural motifs in the protein (such as the RNP consensus sequence) can be analyzed in the living organisms. Immuno-EM with isoform-specific antibodies will indicate whether all forms of the proteins are on the transcripts of a given gene, or whether they bind in a gene-specific manner to RNA molecules. This approach should allow, for the first time, a direct test of the function of these ubiquitous and abundant proteins, whose role in pre mRNA metabolism is poorly-understood and somewhat controversial. Additional experiments are planned to continue EM ultrastructural analysis of the in vivo splicing process.